Metagenomic and culture-dependent analysis of Rhinopithecius bieti gut microbiota and characterization of a novel genus of Sphingobacteriaceae

Culture-dependent and metagenomic binning techniques were employed to gain an insight into the diversification of gut bacteria in Rhinopithecius bieti, a highly endangered primate endemic to China. Our analyses revealed that Bacillota_A and Bacteroidota were the dominant phyla. These two phyla species are rich in carbohydrate active enzymes, which could provide nutrients and energy for their own or hosts’ survival under different circumstances. Among the culturable bacteria, one novel bacterium, designated as WQ 2009T, formed a distinct branch that had a low similarity to the known species in the family Sphingobacteriaceae, based on the phylogenetic analysis of its 16S rRNA gene sequence or phylogenomic analysis. The ANI, dDDH and AAI values between WQ 2009T and its most closely related strains S. kitahiroshimense 10CT, S. pakistanense NCCP-246T and S. faecium DSM 11690T were significantly lower than the accepted cut-off values for microbial species delineation. All results demonstrated that WQ 2009T represent a novel genus, for which names Rhinopithecimicrobium gen. nov. and Rhinopithecimicrobium faecis sp. nov. (Type strain WQ 2009T = CCTCC AA 2021153T = KCTC 82941T) are proposed.

well-being [4][5][6] .Compared with the comprehensive research on human microbiomes, wildlife microbiomes have been less extensively studied.Recent metagenomic analyses by Segal et al. on 180 distinct wild animals across various continents revealed that over 75% of their microbial composition remains uncharacterized.This suggests that wildlife microbiomes possess vast potential as reservoirs for the discovery of novel microbial taxa, genes, enzymes, antimicrobials, and probiotics 7 .Studies on the isolation, identification, and preservation of wildlife microbes not only hold benefits to humans, but also contribute to the conservation of endangered wildlife 8 .
Yunnan snub-nosed monkeys (Rhinopithecus bieti) are highly endangered non-human primates endemic to the Southwest of China 9 .They are the only non-human primates that inhabit in harsh conditions at high altitudes (2600-4600 m), with average annual temperatures ranging from 0.9 to 14.3 °C10-12 .R. bieti prefers eating the beard Lichens (Usnea longissima), which accounts for 60% to 86% of its annual feeding time 10 .Since the inhabitant microbiota could help their hosts to adapt to specific habitats and diets 13 , it is intriguing to know whether commensal microbes play a role in R. bieti's ability to survive and stay healthy in such a hostile plateau habitat.In addition, many studies have shown that gut microbes in herbivorous animals are rich in carbohydrate-active enzymes, which are used to decompose cell wall polysaccharides (such as cellulose, hemicellulose, pectin and lignin) to provide energy and nutrients for themselves or their symbiotic hosts.Therefore, they are important sources for exploring new carbohydrate-active enzymes for industrial and biotechnological applications.Yet, few studies have been performed on wild primates in this regard 14,15 .
The Sphingobacteriaceae family belongs to Bacteroidetes phylum that consists of 14 genera.Among them, Pedobacter, Mucilaginibacter and Sphingobacterium are the three largest genera, accounting for about 89% (217/249) of the validly published new species, and Sphingobacterium is the type genus of this family 16 .The Sphingobacteriaceae family is characterized by the presence of unique sphingolipids in their cell walls and menaquinone 7 as the major respiratory quinone 16 .Members of Sphingobacteriaceae live in a variety of environments, such as guts of mammals, soils, fresh waters, wastewaters, composts, active sludges, and rhizosphere [17][18][19][20][21][22][23] .
In this study, metagenomic analysis was conducted to elucidate the diversity of gut microbes and the profiles of carbohydrate-active enzymes from R. bieti.Culture-dependent analysis was also carried out to investigate the faecal microbes of R. bieti, and a series of new species have been isolated and characterized 24,25 .At present, a bacterium designated as WQ 2009 T was isolated, and the strain represented a novel genus of the family Sphingobacteriaceae based on detailed polyphasic studies.

Isolation, cultivation and preservation
Strain WQ 2009 T was recovered from the faeces of R. bieti collected from the Yunnan Snub-nosed Monkey National Park (27 ○ 39′N 99 ○ 21′E; elevation 3000 m), China.The faecal samples of monkeys were collected from their foraging and resting areas.The local temperature during sampling was around 4-12 °C.Samples were taken within 2 h after defecation.Fresh faeces were collected in 15/50 mL sterilized screw centrifuge tubes according to faecal sizes.The faeces were rinsed with sterile saline (0.9%, w/v) for 3 times, then the surface part was removed with sterilized scalpels and the middle part was left for further experiments.Through serial dilutions with 0.1% sterilized Na 4 P 2 O 7 , samples were spread on Columbia Agar plates (Hopebio, China), and were cultured at 30 °C for at least 7 days.The pure culture was obtained as described previously 24,25 and stored at 4 °C for further study.These pure isolates were cultivated on Columbia or LB agar at 30 °C unless otherwise stated.For long-term storage, bacterial cultures with 20% glycerol (v/v) were maintained at − 80 °C.

Sample preparation and metagenomic sequencing
Faecal samples of R. bieti were collected and handled as described above.For metagenomic sequencing, five tubes of faecal samples were randomly selected and mixed together.The sample was snap-frozen in liquid nitrogen and delivered to QsingKe Biological Technology (Beijing, China) with dry ice for sequencing.
Qualified DNA samples isolated were randomly broken into 350 bp fragments with Covaris ultrasonic breaker (Gene Company Limited, China).The paired-end library was constructed through the steps of DNA ends reparation, 3′-ends A-tailing, adapter ligation, size selection, purification and PCR amplification.The library was qualified by Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System.The qualified library was then sequenced by using Novaseq 6000 sequencing Illumina PE150 platform.

Construction and functional annotation of the gene catalog of the R. bieti
MetaGeneMark v2.10 was used to predict the Open Reading Frames (ORF) from the assembled contigs (≥ 500 bp) obtained above, of which less than 100 nt were filtered out.CD-HIT v4.5.8 32 (parameter: -c 0.95, -G 0, -aS 0.9, -g 1, -d 0) was used to remove the redundant ORFs.Briefly, the ORFs were clustered at 95% identity and 90% coverage, and the longest sequence was selected as the representative gene sequence to create a non-redundant initial gene catalogue.Genes encoding the carbohydrate-active enzymes (CAZymes) were identified and classified based on the CAZymes database by using the carbohydrate-active enzyme analysis toolkit 33 (parameter: E-value = 1 × 10 -5 ).The CAZy results were then analyzed manually to determine the proportion of different CAZymes.DIAMOND 34 was used to map these genes to the sequences of bacteria, fungi, archaea, and viruses from NR database with E-value ≤ 1 × 10 -5 by using blastp, and those with the values ≤ minimum e-value × 10 was selected for further analysis.Finally, clean reads were mapped to the non-redundant ORFs using Bowtie2 v2.4.1 (parameter: -end-to-end, -sensitive, -I 200, -X 400), from which ORFs with alignment count less than 2 were filtered out.And the mapping output was used to calculate the abundance of these ORFs in the sample using the following formula: where r k is the reads count, that is, the number of reads mapped to the k gene, and L K is the gene length, that is, the number of nucleotides in the k gene.The index n represents the set of all genes determined in the catalog, and k is an index indicating a particular gene.

Morphological, physiological and biochemical characterization
Transmission electron microscope (JEM-2100, JEOL, Japan) was applied to observe the cell morphology and measure the cell size of strain WQ 2009 T cultivated on LB agar (Oxoid) at 30 °C for 24 h.The gliding mobility of the cells was determined by using phase-contrast microscopy (Leica DM2000, Wetzlar, Germany).Gram staining was performed by using the classical staining protocols as well as the rapid KOH lysis method 35 .
Hydrolysis of casein, cellulose, Tween 20, Tween 60, Tween 80 and starch was carried out according to the methods described in General and Molecular Microbiology 36 .Catalase activity was assessed by observing whether bubbles formed when a drop of 3% H 2 O 2 was added to freshly cultured cells.API 20NE, API ZYM galleries and Biolog GEN III MicroPlates (bioMérieux) were used for carbon source utilization, and other biochemical and physiological characterization.

Chemotaxonomic analysis
Cells grown on LB agar at 30 °C for 3-7 days were collected and freeze-dried for chemotaxonomic studies.The methyl-estered cellular fatty acids of strain WQ 2009 T were extracted from stationary phase cells as recommended by MIDI technical note 38 , and analysed by using Agilent 7890A GC system according to the standard protocols of the Sherlock Microbial Identification System (version 6.1; MIDI database: RTSBA6).Polar lipids were extracted and analysed by using two-dimensional TLC following protocols reported previously 39 .Respiratory quinones were isolated from freeze-dried cells, and analysed by using HPLC (Agilent 1260) as described 40 .

Phylogenetic and genome analysis
The whole genomic DNA of WQ 2009 T was extracted from mid-log phase cells using the method developed by Andreou 41 .For phylogenetic analysis, the 16S rRNA gene sequence was amplified by using primers 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′) with Super-Fidelity DNA polymerase (Vazyme, China).The amplified fragments were cloned into the pBM16A T-vector and sequenced by TsingKe Biological Technology (Beijing, China).The 16S rRNA gene sequence was also extracted from the genome data of WQ 2009 T using the online ContEst16 tool build-in EzBioCloud 42 .The 16S rRNA gene sequences of the closely related type strains listed in the EzBioCloud database 43 and List of Prokaryotic Names with Standing in Nomenclature 44 were retrieved and aligned with ClustalW 45 , and phylogenetic trees were constructed with neighbor-joining (NJ), maximum-parsimony (MP), and maximum-likelihood (ML) methods using a bootstrap test with 1000 replications in MEGA11 46 .
The whole genome of WQ 2009 T was sequenced using the Illumina NovaSeq PE150 platform at TSINGKE Bioinformatics Technology Co., Ltd (Beijing, China).Clean data was obtained by removing the low-quality data from the raw data with readfq version 10, and assembled with SOAP denovo 2.04, SPAdes, AbySS, and integrated with CISA software.GeneMarkS 47 and tRNAscan-SE program were used to retrieve the related coding genes and the transfer RNA genes, respectively.The assembled genome data was annotated with the prokaryotic genome annotation pipeline in NCBI 48 .Carbohydrate-Active enzymes (CAZymes) were annotated by using the online dbCAN2 meta server 49 , which integrates three tools for automated CAZymes annotation: HMMER,

Overview of the gut microbiota of R. bieti
The resulting clean data generated 138,262 contigs with lengths > 1000 bp for a total length of 494,930,990 bp and N50 of 6082 bp.The obtained contigs were assembled into 196 bins, and after quality control and deduplication, 115 bins were left for subsequent analysis.The 115 non-redundant MAGs (metagenome assembled genomes) comprised 72 high-quality MAGs (≥ 80% completeness, ≤ 10% contamination) and 43 medium-quality MAGs (≥ 50% completeness < 80%, ≤ 10% contamination) (Table S1).The taxonomic annotated result showed that almost all the MAGs could be identified at genus and above levels, but only 13.9% (N = 16) could be identified at the species level, which indicated that the intestine of Yunnan snub-nosed monkey contains abundant unknown microbial resources.Among the 115 annotated MAGs (Fig. 1, Table S2), 114 were classified into 10 bacterial phyla, including Bacillota_A (N = 69), Bacteroidota (N = 21), Bacillota (N = 6), Spirochaetota (N = 5), Verrucomicrobiota (N = 5), Bacillota_C (N = 2), Pseudomonadota (N = 2), Bacillota_C (N = 2), Fibrobacterota (N = 1) and Desulfobacterota (N = 1) and 1 was identified into archaeal phyla, Methanobacteriota (N = 1).Among these, Bacillota_A and Bacteroidota species were also the two most abundant phyla, accounting for 45.2% and 29.8% of the total abundance, respectively (Table S3).This finding is consistent with previous research conducted in humans and animals, because Bacillota_A and Bacteroidota species are known for their ability to metabolize polysaccharides, produce short-chain fatty acids and butyrate, maintain intestinal barrier function, and regulate the immune system, which could contribute to their successful colonization of the gut and establishment of an optimal ecological niche 59,60 .Remarkably, only one archaeal strain, Methanobrevibacter A_smithii, was detected in the intestine of R. bieti, comprising 1.7% of the total abundance.Its significant methane production capacity, coupled with high prevalence, suggests it as the primary methane contributor, offering potential avenues for methane reduction strategies 61 .At the genus level, Cryptobacteroides was the most abundant genus, accounting for 17.1% of the total, followed by CAG-914 (6.0%), CAJOIG01 (3.8%), DTU089 (3.6%), Treponema_D (3.0%), Faecousia (2.9%), et cetera (Fig. 1, Table S3).The bacterial diversity revealed in this study was basically consistent with the previous results obtained by Wu et al., who cloned the 16S rRNA genes of the fecal samples and analyzed the bacterial diversity of R. bieti 15 .More recently, Xia et al. compared the differences in the composition of gut microbiota between wild foraging and diet-provisioned Yunnan snub-nosed monkeys using 16S rRNA gene and metagenomic functional studies, and the main microbial composition was similar to the results of this study 14 .

Diversity profile of CAZymes in the microbiota
Microbes play a pivotal role in regulating matter and energy cycles in natural ecosystems, and are an important source of enzymes in biotechnological and industrial applications.Yunnan snub-nosed monkeys feed on the beard Lichens U. longissimi as their staple food, supplemented by tender leaves and fruits of other plants.It  (Table S4).These CAZymes act synergistically in the breakdown of dietary cellulose, hemicellulose, and pectin to provide energy and nutrients to the gut microbes and their host.The GH families were the most abundant in the metagenomes, including 112 different families, which accounted for 55.4% of the total CAZymes (Table S4).The top 10 abundant families GH2, GH3, GH43, GH13, GH28, GH78, GH77, GH23, GH94 and GH31 accounted for 45.8% of all the GH enzymes.GH2 and GH3 were the two most abundant families accounting for more than 17% of the total GHs, which have broad activities for the synthesis or degrading of oligosaccharides, such as β-galactosidase, β-glucosidase, β-xylosidase, β-manosidase and α-l-arabinofuranosidase. GH13 was also one of the most abundant families, which is more specifically involved in the degradation of starch.Other predicted GH enzymes belonged to cellulases, hemicellulases, debranching enzymes and pectin lyases.
The next abundant CAZymes belonged to GT family (the primary enzymes for carbohydrate synthesis), with 50 families accounting for 26.5% of the CAZymes.Among them, GT2, GT4, GT51, GT35, GT28, GT5 and GT26 accounted for 71.9% of GT family enzymes.These enzymes are responsible for catalyzing the transfer of activated nucleotide sugars to carbohydrates during oligosaccharide biosynthesis 62 .
CBM family was the third most abundant CAZymes, including 54 different families, accounting for 10.5% of the CAZymes.Enzymes of CBM family can enhance the catalytic efficiency of GHs by specifically binding to its substrate and increasing the enzyme concentration 63 .
The 220 PLs were distributed in 16 families, which degrade a variety of uronic acid-containing polysaccharides by β-elimination mechanism 64 .CEs catalyze the de-O or de-N-acylation of substituted saccharides and degrade polysaccharides synergistically with GHs 65 .In this study, 1166 enzymes were predicted as CEs and belonged to 13 families.CE4 (356 genes), CE9 (217 genes) and CE12 (164 genes) were the most abundant families, mainly degrading the acetylated pectin, chitin or xylan.Eight genes fell into the AA10 family (formerly CBM33), which are copper-dependent lytic polysaccharide monooxygenases (LPMOs) that act primarily on recalcitrant polysaccharides, such as chitin and cellulose 66 .The enzymes in AA category being much less than those of other families might be due to the fact that AA enzymes are oxidative enzymes and the gut itself is an anaerobic environment.
Taxonomic profiles of genes encoding CAZymes of GHs, CEs and PLs were also manually analysed.As shown in Fig. 2A, the vast majority (> 84%) of all CAZymes were mainly derived from phylum Firmicutes (52%) and Bacteroidetes (32%).In addition, Firmicutes contributed the most to GHs (52%) and CEs (61%).However, Bacteroidetes was accounted for 32% GHs and 63% PLs (Fig. 2B).Further observations at the levels of family taxonomy (Fig. 2C) revealed that: in the GHs classes, families Bacteroidaceae, Lachnospiraceae, Ruminococcaceae, Clostridiaceae, Prevotellaceae, Bacillaceae, Paenibacillaceae, and Rikenellaceae contributed more than 65%; in  www.nature.com/scientificreports/Bacteria composition of the gut microbiota in pure culture A total of 3065 pure strains were randomly picked out from the faecal samples of R.beiti.After removing duplicated strains based on the characteristics of morphology, color and colony texture, 412 strains were kept for further study.Through 16S rRNA gene sequencing, sequence alignment and inquiry analysis of these strains, 221 actinomycetes and 191 other bacteria were preliminarily identified.The actinomycetes were distributed in 8 orders, 14 families and 25 genera of the class Actinomycetes, with Arthrobacter in Micrococcaceae being the most common species (Table S5).The other 191 bacterial strains were distributed in 4 phyla, including Bacteroidetes, Firmicutes, Proteobacteria and Deinococcus-thermus, which were further divided into 7 classes, 10 orders, 19 families and 30 genera, with Sphingobacterium having the highest number of strains (Table S6).
From the obtained pure cultures, it was found that the similarities of 16S rRNA gene sequences of 20 strains were less than or equal to 98.7% when compared to those of their most related strains (one of the criteria of new species classification) 67 , indicating that these strains were potential new taxa (Table S7).Among them, WQ 047 and WQ 117 have been identified as new taxon and validly published 24,25 , and renamed as Sphingobacterium Rhinopitheci WQ 047 T and Faecalibacter Rhinopitheci WQ117 T , respectively.Among the remaining species, WQ 2009 T has the lowest sequence similarity with its most similar species.Furthermore, WQ 2009 T could not assembled from the faecal metagenome of R.beiti based on binning methodology, indicating that it is a part of the rare biosphere.Therefore, we proposed that it belongs to a new genus of Sphingobacteriaceae family, and its classification was systematically studied in the following work.
Strain WQ 2009 T showed resistance to nalidixic acid, aztreonam, amikacin and vancomycin.The cells were susceptible to ampicillin, cefoperazone, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, gentamicin, kanamycin, norfloxacin, penicillin G, piperacillin and polymyxin B. The morphological and physiological characteristics of strain WQ 2009 T and its most related species in the family Sphingobacteriaceae are summarized in Table 1.

Phylogenetic analysis and genomic characterization
The nearly complete 16S rRNA gene sequence (1491 bp) of strain WQ 2009 T was determined.Comparative sequence analysis of strain WQ 2009 T and the validly published type strains using the EzBioCloud server revealed that the most similar strains were those of the members of family Sphingobacteriaceae.WQ 2009 T showed the highest 16S rRNA similarity to S. kitahiroshimense (94.5%), followed by S. pakistanense and S. faecium at a 94.3% similarity level (Table 3), which was below or near the recommended threshold of 98.7% and 94.5% for differentiation of a new species and a new genus, respectively 68 .Strain WQ 2009 T showed 16S rRNA gene sequence similarities of less than 92.2% to those of the type strains of other 13 genera of family Sphingobacteriaceae.To precisely clarify the taxonomic position of WQ 2009 T , phylogenetic trees were constructed by using the neighborjoining (NJ), maximum-likelihood (ML), and maximum-parsimony (MP) methods based on the most similar 16S rRNA gene sequences of strains from genus Sphingobacterium and representative species from all other 13 genera of the family Sphingobacteriaceae.The maximum-likelihood phylogenetic tree analysis indicated that WQ 2009 T represented a member of the family Sphingobacteriaceae, forming a separate clade within the family Sphingobacteriaceae (Fig. 4).Similar topologies were also confirmed in the neighbor-joining tree (Fig. S2) and the maximum-parsimony tree (Fig. S3), suggesting that WQ 2009 T should be classified as a new genus of the family Sphingobacteriaceae.
The placement of strain WQ 2009 T into a new genus was also supported by the genomic data.The estimated dDDH values between this isolate and S. kitahiroshimense, S. pakistanense and S. faecium were 13.6%, 13.3% and 13.8%, respectively (Table 3), which were far below the commonly used 70% threshold for microbial taxonomy 67 .The calculated ANI values between this isolate and the type strains of its closest taxa were below 71.4%, which was also lower than the threshold (< 74.8%) for genus delineation (Table 3) 69 .Moreover, the AAI values between this isolate and S. kitahiroshimense, S. pakistanense and S. faecium were 64.7%, 64.9% and 65.9%, respectively, which was below or slightly higher than the threshold proposed for a new genus 68 .To confirm the phylogenetic relationship of strain WQ 2009 T , a maximum-likelihood (ML) phylogenomic tree was constructed on the basis of 670 orthologous genes.WQ 2009 T was clearly separated from other genera and formed a distinct branch with a high average branch support of 100% (Fig. S4).Thus, according to the phylogenetic and genomic analysis strain WQ 2009 T deserves a representative of a new genus in the family Sphingobacteriaceae.
The genome of strain WQ 2009 T was 3,144,471 bp with 34 contigs and encoded 2731 genes and 76 tRNAs.The genome size of WQ 2009 T was much smaller than that of closely related strains of genus Sphingobacterium (5.1-6.7 M), but similar to those of genus Albibacterium (3.1 M), Daejeonella (3.4 M), and Solitalea (3.3 M).The G+C content of the genomic DNA was 39.4%.After comparing and annotating the amino acid sequences of these predicted genes with GO, KEGG, COG, NR, Pfam and Swiss-Prot functional databases, it was shown that the number of coding genes was 1854, 2351, 1859, 2444, 1854 and 822, respectively.Totally 279 secreted proteins were predicted by SignalP and TMHMM.When annotated with the Transporter Classification Database, 117 membrane transport proteins were predicted.Six genomic islands were found in the genome of this species, and no prophages or clustered regularly interspaced short palindromic repeat sequences were found.The genome of WQ 2009 T also contained a terpene biosynthetic gene cluster when analysed with antiSMASH.Six genes (GM000637, GM000751, GM001257, GM001350, GM001927 and GM002124) were annotated as potential antibiotic resistant genes.
Bacteroides generally have an abundance of CAZymes, which play a pivotal role in the nutrient-microbiotahost interaction 70,71 .There were 166 potential CAZymes (Fig. S5) in WQ 2009 T when analysed by using the dbCAN server.The signal peptide prediction revealed that 59 of the 166 CAZymes contained signal peptides.These enzymes might be secreted out of the cell or targeted to specific locations in the cell to perform their functions.The 166 CAZymes were divided into five classes: 37 glycosyl transferases (GTs), 80 glycoside hydrolases (GHs), 14 carbohydrate esterases (CEs), 30 carbohydrate binding modules (CBMs) and 5 auxiliary activities (AAs).No polysaccharide lyase (PLs) was found.GHs were the most abundant CAZymes found in WQ 2009 T with 80 genes distributed into 37 different families.The 37 families of GHs comprise a number of enzymes with known activities.These enzymes include α-amylase (EC 3.2.1.1),cellulase (EC 3.3.1.4),lichenase (EC 3.2.1.73),cellobiohydroase (EC 3.2.1.91),xyloglucanendohydrolase (EC 3.2.1.151),α-mannosidase (EC 3.2.1.24),α-fucosidase (EC 3.2.1.51),α-L-rhamnosidase (EC 3.2.1.40),et cetra.The GHs play a key role in carbohydrate metabolism, which hydrolyze complex carbohydrates such as starch, hemicellulose, and cellulose 72 .This was in accordance with the diet of Yunnan snub-nosed monkeys, which mainly live on the lichen plant U. longissima 10,14 .CAZymes from the closely related strains and the type strains of all genus from the family Sphingobacteriaceae were also analysed.All the six classes of CAZymes (GH, GT, CBM, CE, AA, and PL) were found in the genomes of these strains except for strain WQ 2009 T , Albibacterium bauzanese BZ42 T , and Solitalea koreensis DSM 21342 T (Table S8).No PL was found in any of the three strains with much smaller genomes.These results indicated that strains of family Sphingobacteriaceae of phyla Bacteroides were excellent resources for the discovery of new or highly active CAZymes.

Figure 1 .
Figure 1.The taxonomic assignment and abundance distribution of MAGs.

Figure 2 .
Figure 2. Taxonomic assignments for the genes encoding CAZymes.Phylum-level (A) taxonomic assignments for six CAZyme classes GHs, GTs, PLs, CEs, CBMs and AAs; Phylum-(B) and family-level (C) taxonomic assignments for the genes conding for three CAZyme classes GHs, PLs, and CEs.

Figure 3 .
Figure 3.The TEM image of strain WQ 2009 T cultured on LB at 30 °C for 1 day.Bar, 500 nm.

Figure 4 .
Figure 4.The phylogenetic tree based on 16S rRNA gene sequence of strain WQ 2009 T using the maximumlikelihood method.Bootstrap values (expressed as percentages of 1000 replications) of above 50% are shown at branch points.Filobacterium rodentium SMR-C T was used as the outgroup.That is, fewer than 2% alignment gaps, missing data, and ambiguous bases were allowed at any position.Bar, 0.05 substitutions per nucleotide position.